A DNA sequence is described for the gene therapy of diseases of the central nervous system.
The essential elements of this DNA sequence are the activator sequence, the promoter module and the gene for the active substance.
The activator sequence is activated, in a cell-specific manner, in activated endothelial cells or glial cells. This activation is regulated by the promoter module in a cell cycle-specific manner. The active substance is a nerve growth factor, an enzyme of dopanine metabolism and/or a protective factor for nerve cells. The described DNA sequence is inserted into a viral or non-viral vector which is supplemented by a ligand having affinity for the target cell.
After the conclusion of ontogenesis, the nerve cells constitute cells which are fully differentiated and no longer capable of division. In general, they are characterized by the nerve cell body and nerve cell processes, with a distinction being made between afferent (dentrites) and efferent (neurites) processes. The efferent neurite, only one of which is generally formed per nerve cell, makes contact with its target orgen (nerve cells or other types of somatic cell) by way of synapses.
Maintenance of the anatomical structure and function of nerve cells is effected in the presence of neuronal growth factors.
In a general sense, neuronal growth factors are to be understood as being neurotrophic factors (Reviews in Massague, Cell 49, 437 (1987), Pusztai et al., J. Pathol. 169, 191 (1993), Ibanez et al., PNAS 89, 3060 (1992), Sonoda et al., BBRC 185, 103 (1992)). These factors include neuronal growth factors in Table 1.
In a narrower sense, the nerve growth factor (NGF) family should be included in these factors.
NGFs act by way of binding to NGF receptors, which are formed, in particular, on the sensory nerve fibers. NGF is taken up intracellularly and transported to the nerve cell body in a retrograde manner (Johnson et al., J. Neurosci. 7, 923 (1987)). In the nerve cell body, the NGF probably brings about an increase in cyclic adenosine monophosphate (cAMP), with subsequently elevated efflux of Ca++ (Schubert et al., Nature 273, 718 (1978), and, in addition, release of diacylglycerol and activation of protein kinase C, by way of inositol lipid metabolism, and intracellular release of CA++ by way of inositol triphosphate liberation (Abdel-Latif, Pharmacol. Rev. 38, 227 (1986)).
The phosphorylation of specific, in particular signal-transducing, proteins which results from this leads to changes in their function. This results in an increased formation of proteins which are involved in the growth of neurites. These proteins include chartin proteins (Black et al., J. Cell Biol. 103, 545 (1986)) tau proteins and tubulins (Drubin et al., J. Cell Biol. 101, 1799 (1985)). Thus, the synthesis of xcex1-tubulins and xcex2-tubulins, neuro-filament proteins (NF-L, NF-M and NF-H) and peripherin (Portier et al., Devi Neuroscience 6, 215 (1983)), Parysek et al., J Neurosci. 7, 78, (1987)) is elevated. At the same time, the concentration of enzymes which are important in the nervous system, such as choline acetyl-transferase, acetylcholinesterase and neurone-specific enolase (Vinores et al., J. Neurochemistry 37, 597, 1981), Rydel et al., J. Neurosci. 7, 3639 (1987)) increases.
In addition, the concentrations of neurotransmitters, such as neurotensin (Tischler et al., Reg. Pept. 3, 415 (1982)) and neuropeptide Y (Allen et al., Neurosci. Lett. 46, 291 (1984)), and neurotransmitter receptors, such as acetylcholine receptors (Mitsuka et al., Brain Res. 314, 255 (1984)) and encephalin receptors (Inoue et al., J. Biol. Chem. 257, 9238 (1982)) are increased. At the same time, the concentration of synapsin 1 is increased (Romano et al., J. Neurosci. 7, 1300 (1987)).
In the final analysis, NGF maintains the functional state of nerve cells. At the same time, NGF initiates and promotes the growth of neurites. The constant presence of NGF is necessary for this neuritogenic and synaptogenic activity (Smith, Science 242, 708 (1988), Mitchison et al., Neuron 1, 761 (1988)).
This has been reported, in particular, for ciliary neurotrophic factor (CNTF) (Lin et al., Drugs of the Future 19, 557 (1994)).
The neurotrophic activity of neuronal growth factors has been substantiated experimentally, in particular in association with damage to nerve cells, for example in association with the surgical severence of neurites. If CNTF is administered locally to the proximal stump of the transected nerves, the proportion of nerve cells which die following the surgical intervention is markedly reduced (Sendtner et al., Nature 345, 440 (1990)). At the same time, the concentration of, for example, the neuro-peptide substance P is markedly elevated in the spinal ganglia following CNTF administration. Rats whose sciatic nerve has been damaged exhibit accelerated restoration of the motor activity following subcutaneous administration of CNTF (Lin et al., Drugs of the Future 19, 557 (1994)).
However, systemic administration of neurotrophic factors is only effective if the motor neurones, which are present in the spinal cord and which are protected by the blood-brain barrier, possess axons which are still functional outside these barriers and by way of which the neurotrophic factors can be taken up (Apfel et al., Brain Res. 605 1 (1993)).
In the case of nerve cell damage up to the other side, or on the other side, of the blood-brain barrier, it is necessary to administer neurotrophic factors intracranially. In this way, retrograde generation of the proximal thalamic neurones following severance of the thalamic axons can, for example, be prevented experimentally (Clatterbuch et al., PNAS 90, 2222 (1993)). However, a prerequisite for an optimal regeneration process is the constant presence of the neutrophic factors at the site of the damaged nerve cell. While it is possible to effect a local administration at the time of the surgical damage or damage relief, this is difficult, or almost impossible to do once the surgical intervention has ended. Diffuse damage to the CNS, for example due to blunt trauma or toxins, affords only very limited opportunity for effecting a targeted administration.
Glial cells can be stimulated to produce TNF as a result of traumatic, immunological and toxic influences. This TNF xcex1 is, at that time, toxic for nerve cells and glial cells, (Owens et al., Immunol. Today 15, 566 (1994)).
In order to achieve a presence of active compounds in the CNS which is as long-term as possible, attempts are made to inject intracranially cells (fibroblasts, endothelial cells and myoblasts) which have been transduced in vitro to express neurotrophic active compounds. The aim is to use the neurotrophic active compounds to improve the regeneration and function of nerve cells which have been damaged traumatically or degeneratively, for example in Parkinson""s disease or in dementia. Especially in the case of Parkinson""s disease, attempts are made to inject cells, which either have been transduced in vitro, to secrete neurospecific enzymes such as tyrosine hydroxylase and dopa decarboxylase (Kopin, Ann. Rev. Pharmacol. Toxicol. 32, 467 (1993), Fisher et al., Physiol. Rev. 11, 582 (1993), Jiao et al., Nature 362, 450 (1993)), or else human fetal, dopaminergic, nigral neurones are injected (Lxc3x6wenstein, Bio/Technology 12, 1075 (1994)).
However, cells of this nature are only available in limited quantities. On the other hand, the use of fetal cells raises important ethical questions.
As an alternative, the possibility is being examined of injecting vectors directly into the brain in order to transduce brain cells to express the desired active compounds (During et al., Science 266, 1399 (1994)). However since these vectors do not exhibit any cell specificity, there is the substantial risk of nerve cells being damaged by infection or transfection with the vector.